ABSTRACT: The 3' untranslated regions (3' UTRs) of mRNAs contain cis-acting elements for posttranscriptional regulation of gene expression. Here, we report that mouse genes tend to express mRNAs with longer 3' UTRs as embryonic development progresses. This global regulation is controlled by alternative polyadenylation and coordinates with initiation of organogenesis and aspects of embryonic development, including morphogenesis, differentiation, and proliferation. Using myogenesis of C2C12 myoblast cells as a model, we recapitulated this process in vitro and found that 3' UTR lengthening is likely caused by weakening of mRNA polyadenylation activity. Because alternative 3' UTR sequences are typically longer and have higher AU content than constitutive ones, our results suggest that lengthening of 3' UTR can significantly augment posttranscriptional control of gene expression during embryonic development, such as microRNA-mediated regulation. Overall design: Two biological replicates of C2C12 growth and differentiation conditions, repesctively

Similar Datasets

Project description:The 3' untranslated regions (3' UTRs) of mRNAs contain cis-acting elements for posttranscriptional regulation of gene expression. Here, we report that mouse genes tend to express mRNAs with longer 3' UTRs as embryonic development progresses. This global regulation is controlled by alternative polyadenylation and coordinates with initiation of organogenesis and aspects of embryonic development, including morphogenesis, differentiation, and proliferation. Using myogenesis of C2C12 myoblast cells as a model, we recapitulated this process in vitro and found that 3' UTR lengthening is likely caused by weakening of mRNA polyadenylation activity. Because alternative 3' UTR sequences are typically longer and have higher AU content than constitutive ones, our results suggest that lengthening of 3' UTR can significantly augment posttranscriptional control of gene expression during embryonic development, such as microRNA-mediated regulation. Two biological replicates of C2C12 growth and differentiation conditions, repesctively

Project description:Much of posttranscriptional mRNA regulation occurs through cis-acting sequences in mRNA 3´ untranslated regions (UTRs), which interact with specific proteins and ribonucleoprotein complexes that modulate translation, mRNA stability and subcellular localization. Studies in Caenorhabditis elegans have revealed indispensable roles for 3´UTR-mediated gene regulation, yet most C. elegans genes have lacked annotated 3´UTRs. Here we describe a high-throughput method to reliably identify 3´ ends of polyadenylated RNAs. This method, called poly(A)-position profiling by sequencing (3P-Seq), was used to determine the UTRs of C. elegans. Compared to standard methods also recently applied to C. elegans UTRs, 3P-Seq identified 8775 additional UTRs while excluding thousands of shorter UTR isoforms that do not appear to be authentic. Analysis of this expanded and corrected dataset indicated that the high A/U content of C. elegans 3´UTRs facilitated genome compaction, since the elements specifying cleavage and polyadenylation, which are A/U-rich, can more readily emerge in A/U rich regions. Indeed, 30% of the protein-coding genes have mRNAs with alternative, partially overlapping end regions that generate another 10,000 cleavage and polyadenylation sites that had gone largely unnoticed and represent potential evolutionary intermediates of progressive UTR shortening. Moreover, a third of the convergently transcribed genes utilize palindromic arrangements of bidirectional elements to specify UTRs with convergent overlap, which also contributes to genome compaction by eliminating regions between genes. Although nematode 3´UTRs have median length only one-sixth that of mammalian 3´UTRs, they have twice the density of conserved microRNA sites, in part because additional types of seed-complementary sites are preferentially conserved. These findings reveal the influence of cleavage and polyadenylation on the evolution of genome architecture and provide resources for studying posttranscriptional gene regulation. Overall design: Nine samples (10 sequencing runs) from various mixed and specific stages of wild-type Caenorhabditis elegans and glp-4 mutant adults.

Project description:Much of posttranscriptional mRNA regulation occurs through cis-acting sequences in mRNA 3´ untranslated regions (UTRs), which interact with specific proteins and ribonucleoprotein complexes that modulate translation, mRNA stability and subcellular localization. Studies in Caenorhabditis elegans have revealed indispensable roles for 3´UTR-mediated gene regulation, yet most C. elegans genes have lacked annotated 3´UTRs. Here we describe a high-throughput method to reliably identify 3´ ends of polyadenylated RNAs. This method, called poly(A)-position profiling by sequencing (3P-Seq), was used to determine the UTRs of C. elegans. Compared to standard methods also recently applied to C. elegans UTRs, 3P-Seq identified 8775 additional UTRs while excluding thousands of shorter UTR isoforms that do not appear to be authentic. Analysis of this expanded and corrected dataset indicated that the high A/U content of C. elegans 3´UTRs facilitated genome compaction, since the elements specifying cleavage and polyadenylation, which are A/U-rich, can more readily emerge in A/U rich regions. Indeed, 30% of the protein-coding genes have mRNAs with alternative, partially overlapping end regions that generate another 10,000 cleavage and polyadenylation sites that had gone largely unnoticed and represent potential evolutionary intermediates of progressive UTR shortening. Moreover, a third of the convergently transcribed genes utilize palindromic arrangements of bidirectional elements to specify UTRs with convergent overlap, which also contributes to genome compaction by eliminating regions between genes. Although nematode 3´UTRs have median length only one-sixth that of mammalian 3´UTRs, they have twice the density of conserved microRNA sites, in part because additional types of seed-complementary sites are preferentially conserved. These findings reveal the influence of cleavage and polyadenylation on the evolution of genome architecture and provide resources for studying posttranscriptional gene regulation. Nine samples (10 sequencing runs) from various mixed and specific stages of wild-type Caenorhabditis elegans and glp-4 mutant adults.

Project description:The 3' untranslated regions (3' UTRs) of mRNAs play important roles in the regulation of mRNA localization, translation, and stability. Alternative cleavage and polyadenylation (APA) generates mRNAs with different 3' UTRs, but the involvement of this process in stress response has not been clarified. Here, we report that a subset of stress-related genes exhibits 3' UTR extensions of their mRNAs during dehydration stress. These extended 3' UTRs have characteristics of long noncoding RNAs and likely do not interact with miRNAs. Functional studies using T-DNA insertion mutants reveal that they can function as antisense transcripts to repress expression levels of sense genes from the opposite strand, or can activate the transcription of downstream genes from the same strand. Poly(A) signal analysis reveal that mRNAs with 3' UTR extensions have weaker poly(A) signals than mRNAs without 3' UTR extensions. Finally, we show that their biogenesis is partially dependent on a trans-acting factor FPA. Taken together, we report that dehydration stress could induce mRNA 3' UTR extensions, and elucidate a novel function for these stress-induced 3' UTR extensions as long noncoding RNAs in the regulation of their neighboring genes. Overall design: To study the effects of stress on APA, we performed ssRNA-seq on Arabidopsis seedlings grown in normal, ABA-treated, and dehydration-stressed conditions. We collected samples at two time points (1 h and 3 h) for each treatment and for each time point we had three biological replicates.

Project description:The proper subcellular localization of RNAs and local translational regulation is crucial in highly compartmentalized cells, such as neurons. RNA localization is mediated by specific cis-regulatory elements usually found in mRNA 3'UTRs. Therefore, processes that generate alternative 3'UTRs – alternative splicing and polyadenylation – have the potential to diversify mRNA localization patterns in neurons. Here, we performed mapping of alternative 3'UTRs in neurites and soma isolated from mESC-derived neurons. Our analysis identified 593 genes with differentially localized 3'UTR isoforms. In particular, we have shown that two isoforms of Cdc42 gene with distinct functions in neuronal polarity are differentially localized between neurites and soma of mESC-derived and mouse primary cortical neurons, at both mRNA and protein level. Using reporter assays and 3'UTR swapping experiments, we have identified the role of alternative 3’UTRs and mRNA transport in differential localization of alternative CDC42 protein isoforms. Moreover, we used SILAC to identify isoform-specific Cdc42 3'UTR-bound proteome with potential role in Cdc42 localization and translation. Our analysis points to usage of alternative 3'UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms.

Project description:Most eukaryotic genes express alternative polyadenylation (APA) isoforms with different lengths of 3’ untranslated region (3’UTR). Here we show arsenic stress elicits global shortening of 3’UTRs through two mechanisms. First, stress leads to immediate shortening of 3’UTR due to preferential usage of proximal cleavage and polyadenylation sites (PASs), as revealed by 3’ end sequencing of newly made RNAs that are metabolically labeled with 4-thiouridine. Second, long 3’UTR isoforms are more rapidly degraded during recovery from stress as compared to short 3’UTR isoforms, further shortening 3’UTR lengths in the cell. Using ribonucleoprotein immunoprecipitation coupled with 3’ end sequencing (3’READS+RIP), we show that the RNA-binding protein T cell-restricted intracellular antigen-1 (Tia1) preferentially interacts with long 3’UTR isoforms via U-rich elements in alternative 3’UTR sequences, and the interaction correlates with stress granule (SG) association during stress and with mRNA decay during recovery from stress, indicating SG-mediated RNA clearance mechanism post stress. Importantly, genes whose 3’UTRs are shortened by APA during stress can evade stress-induced 3’UTR size-based mRNA degradation, leading to higher transcript abundance post stress. Moreover, proliferating and differentiated cells display different extents of 3’UTR shortening after stress, indicating cell type-specific of impact of stress on the 3’UTR landscape. Together, our data indicate that 3’UTR length plays important roles in gene expression in stressed cells, and APA functions as an adaptive stress response mechanism to preserve mRNAs. Overall design: 4 RNA-seq libraries from NIH3T3 cells for gene expression analysis; 4 3'READS libraries for analysis of APA using total RNA from NIH3T3 cells treated with sodium arsenite for 1h; 6 3'READS libraries for analysis of APA time course using total RNA from NIH3T3 cells treated with sodium arsenite for 1h and recovered for different amount of time; 8 3'READS libraries for analysis of APA isoform stability using 4sU metabolic labeling in NIH3T3 cells; 8 3'READS libraries for analysis of stress response of proliferating and differented C2C12 cells using cytoplasmic RNA; 4 3'READS libraries for the analysis of APA during C2C12 cell differentiation.

Project description:N6-methyadenosine (m6A) is enriched in 3`UTR (3` untranslated region) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. Herein we report a characterization of the full m6A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and we show that VIRMA mediates preferential mRNA methylation in 3`UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m6A enrichment in 3`UTR. Depletions of VIRMA and METTL3 induce 3`UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3`UTR of over 2,800 mRNAs, 84% of which are modified with m6A. Together our studies provide insights into m6A deposition specificity in 3`UTR and its correlation with alternative polyadenylation. Overall design: We used VIRMA KO/KD cell lines to test m6A deposition specificity in 3`UTR and its correlation with alternative polyadenylation.

Project description:Messenger RNA stability, localization, and translation are largely determined by sequences in their 3′ untranslated regions (3’UTRs), which recruit regulatory proteins and RNAs. More than half of the mammalian genes generate multiple mRNA isoforms differing in their 3′UTRs and therefore in their regulatory elements. The Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1) binds to cognate sites in 3’ UTRs and regulates translation. CPEB1 can shuttle to the nucleus and we report its co-localization with splicing factors. CPEB1 knock down leads to changes in alternative splicing, and we show that alternative 3’ splice site linked to alternative polyadenylation of the bub-3 pre-mRNA, important for cell proliferation, is regulated by CPEB1 at least in part by preventing 3’ splice site recognition by U2AF. RNA-Seq experiments reveal that CPEB1 mediates 3’ UTR shortening of hundreds of mRNAs, leading to changes in their translation efficiency. Three total RNA from three biological replicates were labeled and hybridized versus its own control in direct and dye-swap hybridizations.

Project description:In eukaryotes, the 3' ends of RNA polymerase II-generated transcripts are made in the majority of cases by site-specific endonucleolytic cleavage, followed by the addition of a poly(A) tail. By alternative polyadenylation, a gene can give rise to multiple mRNA isoforms that differ in the length of their 3' UTRs and hence in their susceptibility to post-transcriptional regulatory factors such as microRNAs. A series of recently conducted high-throughput studies of poly(A) site usage revealed an extensive tissue-specific control of 3’ UTR length and drastic changes in 3’ UTR length of mRNAs upon induction of proliferation in resting cells. To understand the dynamics of polyadenylation site usage, we recently identified binding sites of the major pre-mRNA 3’ end processing factors - cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and cleavage factor Im (CF Im) - and mapped cleaved polyadenylation sites in HEK293 cells. Our present study extends previous findings on the role of CF Im in alternative polyadenylation and reveals that subunits of the CF Im complex generally control 3’ UTR length. More specifically, we demonstrate that the loss-of-function of CF Im68 and CF Im25 but not of CF Im59 leads to a transcriptome-wide increase of the use of proximal polyadenylation sites. 3' ends of transcripts were profiled by high-throughput sequencing in HEK 293 cells under normal conditions, and in HEK 293 cells depleted of 3' end processing factors CF Im25, CF Im59, and CF Im68.